Mannose-binding lectin (“MBL”) is a mammalian serum protein which is involved in innate immunity against microbial infections. MBL recognizes the specific glycosylation patterns of the proteins on the cell surface of the infecting microorganism and binds to them to suppress the microbial infection according to three pathways described below. In the first pathway, MBL binds the microbial glycosylated protein to form a complex, and then activates MBL associated serine proteases (“MASPs”). The MASPs, in turn, cleave the second and fourth complement components (“C4” and “C2”), leading to an activation of the complement system. In the second pathway, MBL, bound to the glycosylated microbial cell surface proteins, serves as an opsonin and directs phagocytosis by neutrophils and macrophages. The same bound MBL can also neutralize the infectivity of the microbes in the third pathway, blocking their proliferation. Thus, the most important initial step in the MBL's defense against microbial infections is the recognition of and binding to the microbes.
MBL, a member of the collectin family, shares a common structure consisting of a collagen domain and a carbohydrate recognition domain (lectin domain) with other members of the family. The MBL monomer has a molecular weight of 32 kDa. It has a C-type carbohydrate recognition domain (“CRD”) at the C-terminus, a cysteine-rich region at the N-terminus, and a collagen domain in-between. Three identical MBL monomeric polypeptides associate to form a triple helical complex through their collagen domains. By the disulfide bond formation of cysteines in the N-terminal region, up to six of the triple helical complex form oligomeric flower bouquet like molecules, consisting of dimmers, trimers, and so on to hexamers. This triple helix is a structural feature shared by all the collectin family members including surfactant proteins A (“SP-A”) and D (“SP-D”), collectin-liver 1 (“CL-L1”), and collectin-placenta 1 (“CL-P1”). Thus, these member proteins belonging to the collectin family all share similar physicochemical properties. These collectin family proteins have been known to share another characteristic of playing an important role in the pre-immune defense against microbial infections in sera and the pulmonary surface as well (Hans-Jurgen et al, Protein Science, 3:1143, 1994). Besides the ones listed above, other family members include collectin-43 (“CL-43”), collectin-46 (“CL-46”), a bovine conglutinin, and a human conglutinin homolog.
MBL can bind to a wide range of oligosaccharides. As the target sugars are not normally exposed on mammalian cell surfaces at high densities, MBL does not usually recognize self-determinants, but is particularly well suited to interactions with microbial cell surfaces presenting repetitive carbohydrate determinants. MBL most often binds viruses with outer coats (viral envelopes). Representative examples include: influenza virus (Hartshorn, K. L. et al., J. Clin. Invest., 91:1414, 1993; Kase, T. et al., Immunol., 97:385, 1999), human immunodeficiency virus (“HIV”) (Ezekowitz, R. A. et al, J. Exp. Med., 169:185, 1989; Haurum, J. C. et al., AIDS, 7:1307, 1993), herpes virus (Fischer, C. B. et al., Scan. J. Immunol., 39:439, 1994), and SARS corona virus (Ksiazek, T. G. et al., N. Eng. J. Med., 348:1953, 2003; Peiris, J. S. M. et al., Lancet, 361:1319, 2003). Rhinoviruses, responsible for the common cold, are expected to be good binders to MBL as well. Among bacteria, Staphylococcus aureus (Neth, O. et al., Infect. Immunol., 68:688, 2000) and Hemophilus influenzae (Van E. et al., Clin. Exp. Immunol., 97:411, 1994) are reported to be good binders, whereas among fungi, Candida albicans (Tabona, P. et al., Immunol., 85:153, 1995) is known to bind MBL.
Among the microbes that bind MBL, influenza virus, rhinovirus, severe acute respiratory syndrome (“SARS”), corona virus, and influenza virus of animal origin cause symptoms mainly through the infection of the respiratory epithelia, whereas S. aureus and H. influenzae infect lungs. S. aureus can also infect external wounds and C. albicans is involved in vaginitis. Since treating MBL in a solution phase is not suitable for curing these respiratory diseases and external wounds, special formulations are required. Although there have been many studies on MBL, the extent to which MBL is involved in the defense against the infections in epithelia and external wounds mentioned above is poorly understood. The only relevant study is one reporting a detection of MBL in saliva and breast milk (Tregoat, V. et al., J. Clin. Lab. Anal., 16(6): 304, 2002).
Upon influenza virus infection, the virus proliferates in the epithelial cells that line the surfaces of respiratory organs. Once the virus has amplified itself inside the infected cell, new virus released from the cell infects neighboring epithelial cells. Thus, it is possible to disrupt contact between an epithelial cell and the virus using MBL which is capable of recognizing the glycosylated microbial surface proteins and binding to them. Thus, MBL binding to glycosylated flu virus surface protein, haemagglutinin and neuraminidase, blocks the new virus infection to the neighboring epithelial cells. In fact, it was observed in cultured cells that physical neutralization by MBL binding was sufficient for blocking microbial infection of neighboring cells. For example, MBL added to the culture medium prevented viral infection upon SARS corona virus inoculation of cultured cells (Korean patent gazette No. 1020040106194). Similar prevention of infection by MBL has been observed for influenza virus as well (Wakamiya, N. et al., Biochem. Biophys. Res. Commun., 187:1270, 1992; Hartley, C. A. et al, J. Virol., 66:4358, 1992; Patrick, C. R. et al., J. Virol., 71:8204, 1997; Kase, T. et al., Immunol., 97:385, 1999).
In order to treat external wounds and respiratory infections with MBL, it is desirable in many ways to formulate the protein into a powder. A powder formulation is capable of an effective delivery to sites of infection, delivers an optimal amount of MBL, lessens side effects due to its topical application, and reduces the amount used.
Freeze-drying and spray-drying are used in general to formulate protein drugs into powders. Freeze-drying is most widely employed nowadays. Freeze-drying is suitable for heat-sensitive proteins, but it is not suitable for producing a uniform powder with a diameter of a few micrometers, a form that can be readily inhaled. Freeze-drying also tends to concentrate proteins locally between the ice crystals while cooling. This local concentration brings about a rapid change in the pH and ionic strength surrounding the protein to cause protein denaturation and precipitation (Schwartz, P. L. et al., Endocrinology, 92(6): 1795, 1973; Koseki, T. et al., J. Biochem., 107:389, 1990). In contrast, spray-drying involves spraying a continuous stream of a liquid sample to form microscopically dispersed droplets, while instantaneously drying them with hot air at the same time. Spray-drying has been in use for formulating various drugs. Spray-drying has the advantage of producing powders whose particle sizes are suitable for delivering drugs to respiratory tracts and lungs. Spray-drying also consumes less energy so that time and cost can be saved at the production line (Broadhead, J. et al., Drug Dev. Ind. Pharm., 18(11&12):1169, 1992). Since proteins in general are not stable against heat, there are not many cases of protein drug formulations spray-dried with hot air. There are attempts, however, to apply spray-drying as follows: oxyhemoglobin (Labrude, P. et al., J. Pharm. Sci., 78(3):223, 1989), human growth hormone (Mumenthaler, M. et al., Pharm. Res., 11(1):12, 1994; Bosquillon, C., J. Cont. Rel., 96:233, 2004), tissue plasminogen activator (Mumenthaler. M. et al., Pharm. Res., 11(1): 12, 1994), DNase (Chan, H. K. et al., Pharm. Res., 14(4):431, 1997), parathyroid hormone (Codrons, V. et al., J. Pharm. Sci., 92(5):938, 2003), and humanized monoclonal antibody, anti-IgE, (Maa, Y. F. et al., Biotechnol. Bioeng., 60(3):301, 1998).
Especially when producing spray-dried protein compositions for respiratory applications, it is desirable to formulate proteins into powders with particle sizes of 5 μm or less. The particle size, shape and water content of such powders are important factors in terms of treatment efficacy (Hickey, A. J. et al., Pharm. Tech., 18:58, 1994). The major determinants of the physical characteristics of such powders are mechanical conditions such as the feed velocities for hot air and the protein solution, temperature and spray pressure (Maa, Y. F. et al., Pharm. Res., 15(5):768, 1998) as well as the identities and concentrations of the excipients to the protein solution such as salts, sugars, and proteins (Andya, J. D. et al., Pharm. Res., 16(3):350, 1999; Maa, Y. F. et al., Pharm. Dev. Tech., 2(3):213, 1997). Also in the dry powder formulation of recombinant human MBL by spray-drying, the important considerations are that a spray-dried MBL powder composition should maintain its ability to activate complement through the specific binding to the glycosylated, MBL-binding proteins in the presence of serine proteases when dissolved and that such ability is also not lost during long-term storage.
However, there have been no studies reported on spray-drying methods for producing powder collectin composition in general, human recombinant MBL compositions in particular, for treating illnesses such as respiratory inflammation. Thus, there is a strong need for spray-drying methods to produce a dry powder collectin composition available for respiratory inhalation and application to external wounds. Accordingly, the present invention provides a dry powder collectin composition formed by adding a carbohydrate and/or protein excipients to a solution containing at least one collectin family member protein and spray-drying the solution. This powder is able to support long-term storage without losing its efficacy.